The present invention relates to a basic protein isolated from the venom of a snake of the family Elapidae, especially of a Naja snake and more particularly Naja mossambica pallida and/or Naja nigricollis, the said protein being called phospholipase A2 (PLA2), to fragments and derivatives of the said protein, to their methods of preparation, to therapeutic compositions which can be used in human and/or veterinary medicine and to diagnostic agents in which the said protein and/or its derivatives and/or its fragments are present.
Numerous antitumoral agents, which are either organic or inorganic substances, exist at the present time. These various agents act on the cell nucleus, in particular the nucleus of tumoral cells. There are several mechanisms of action according to the substance in question: either the said substances interfere with the biosynthesis of the nucleic acids and the proteins, or they interfere with the duplication of the DNA, or they act on the mitotic spindle. A number of authors have demonstrated these different mechanisms of action (R. K. RALPH et al., TIBS, 1983, 212-214; LIPPARD, Science, 1982, 218, 4577, 1075-1082; FAUVAUDON, Biochimie, 1982, 64, 457-475).
However, anticancer substances do not exert their action specifically on cancerous cells. They inhibit the division of healthy cells as well as cancerous cells, so they are toxic to all tissues in which the cells renew themselves rapidly. The various antitumoral substances referred to therefore have a large number of undesirable effects in man.
The magnitude of the undesirable effects has led to the search for new antitumoral agents which have a different mode and site of action and which do not have undesirable effects such as those encountered with the existing antitumoral substances.
It has recently been shown that certain substances secreted by cells of various lines, such as, in particular, B and T lymphocytes as well as macrophages (Ruddle, Immunol. Today, 1987, 8, 5, 129-130), are capable of killing the cells of certain tumoral lines. The genes (cDNA) coding for these substances have been cloned (GRAY et al., Nature, 1984, 312, 721-724; PENNICA et al., Nature, 1984, 312, 724-727). These substances are known as tumor necrosis factors (TNF) and lymphotoxins (LT); it should be noted that the mechanism of action of these substances is not known at the present time.
There is another extremely valuable group of substances, which are substances present in snake venoms; the latter are in fact capable of killing cancerous cells of numerous lines (CHAIM-MAYAS et al., Life Sciences, 1987, 40, 16, 1601-1607); the authors of this publication have shown in particular that snake venoms have a cytotoxic activity towards certain melanomas and chondrosarcomas, both in vitro and in vivo, and especially that the venom of Naja nigricollis has a high cytotoxic activity.
This cytotoxic activity seems to be based on a mechanism of membrane lysis of the cells in question which is totally different from the mechanisms of action of the antitumoral substances of the prior art.
The venom of the spitting cobra of East Africa, Naja nigricollis, like the venom of the majority of the Elapidae, contains three classes of toxic protein: curarizing toxins, cardiotoxins and phospholipases.
The cardiotoxin, which is the preponderant component of the venom, is responsible inter alia for the cytotoxic action; however, the cytotoxic activity of the total venom is very much greater than that of the cardiotoxin alone. Moreover, the cytotoxic action of the venom is retained in the presence of calcium, which is an inhibitor of the cytotoxic activity of cardiotoxins. One or more cytotoxic factors other than the cardiotoxin are therefore present in the venom of Naja, in particular Naja nigricollis.
The phospholipases are classed in different groups according to their lethality. The first group includes acidic or neutral phospholipase A2's, which have a low toxicity; these PLA2's essentially ensure digestion of the phospholipidic tissues. The second group is made up of extremely toxic PLA2's; these are either monomeric basic molecules or molecules containing a basic subunit. These compounds are found in numerous snake venoms and cause rapid paralysis of the prey. They are characterized by an ability to block the release of acetylcholine at the presynaptic nerve endings and by other effects, including substantial myotoxicity in particular. The third group comprises strongly basic, toxic PLA2's; these are found in a number of snake venoms, but their role and their mechanism of action were not well understood until now; the basic PLA2 of Naja nigricollis belongs to this category.